Over 50% of patients with advanced Oral cavity Squamous Cell Carcinomas (OSCCs) seen at major tertiary centers die within five years of diagnosis following relapse from frontline therapy. We believe that improvements to overall survival will be made by improving our ongoing precision medicine trials, in which the right therapies are given at the right time, but this requires a comprehensive understanding of molecular subsets of the disease. Despite the knowledge that has been gained from publicly available OSCC sequencing data and because of the diversity of drivers and lost suppressors, too few cases have been sequenced to molecularly stratify the disease on an integrated genomic and transcriptomic level. Importantly, several targeted therapies have been advanced for common molecular alterations including EGFR, FGFR1/3, PIK3CA, NOTCH, etc.; however, these often perform poorly as monotherapies due to innate genetic or compensatory resistance creating a strong need for rational combination therapy. In fact, various members of the NOTCH pathway are mutated in ~50% of all OSCCs, and while we and others have recently advanced WNT pathway inhibitors to clinical trials for this molecular subset, NOTCH-deficient tumors frequently harbor additional lesions that can confound the therapeutic benefits of targeted monotherapy. We have collected a unique set of surgically treated responsive and relapsed OSCC tumors, which we propose to study for the frequency of co-altered lesions using integrative genetic and transcriptomic sequencing. Through preliminary pooled CRISPR and small molecule screening of genetically defined OSCC cell line models, we will validate a strategy that defines targets for combination therapy. Further, we show preliminary results using surgically excised, ex vivo OSCC tissue as a model system to evaluate novel combination therapies. Leveraging these tools and techniques, we will test our central hypothesis that innate or compensatory pathways, which drive resistance to targeted monotherapies, can be identified through our integrative approach and exploited to develop effective combination protocols that overcome resistance. We will address this hypothesis with the following Aims: 1) Identify co-dependent molecular targets for combination therapy through integrative next generation sequencing in surgically non-responsive OSCC, 2) Define the combinations of genes and pathways pivotal for cell proliferation in OSCC cell lines using lentiviral CRISPR and small molecule libraries, 3) Develop combination treatment strategies for genetically defined OSCC using in vivo models. Our primary goal is to develop novel combination strategies to improve the survival of patients with OSCC through identification of co-dependent therapeutic targets.